WO2019191133A1 - Surveillance en temps réel de la concentration de protéines à l'aide d'un signal ultraviolet - Google Patents

Surveillance en temps réel de la concentration de protéines à l'aide d'un signal ultraviolet Download PDF

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Publication number
WO2019191133A1
WO2019191133A1 PCT/US2019/024132 US2019024132W WO2019191133A1 WO 2019191133 A1 WO2019191133 A1 WO 2019191133A1 US 2019024132 W US2019024132 W US 2019024132W WO 2019191133 A1 WO2019191133 A1 WO 2019191133A1
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WIPO (PCT)
Prior art keywords
titer
protein
target protein
cells
psi
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PCT/US2019/024132
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English (en)
Inventor
Deqiang Yu
Xiaoming Liu
Chao Ma
Jongchan Lee
Zhengjian Li
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Bristol-Myers Squibb Company
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Application filed by Bristol-Myers Squibb Company filed Critical Bristol-Myers Squibb Company
Priority to JP2020551818A priority Critical patent/JP2021519298A/ja
Priority to EP19719664.5A priority patent/EP3774840A1/fr
Priority to US17/041,851 priority patent/US20210024873A1/en
Priority to KR1020207030632A priority patent/KR20200136454A/ko
Priority to CN201980022884.1A priority patent/CN111918873A/zh
Publication of WO2019191133A1 publication Critical patent/WO2019191133A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/79—Photometric titration

Definitions

  • the present disclosure is related to a method of monitoring concentration of a biological molecule, e.g., a protein, in a composition. Specifically, the present disclosure is directed to a method of monitoring, controlling, modulating, or increasing protein yield from a composition using real-time ultraviolet signal during protein filtration.
  • a biological molecule e.g., a protein
  • mAbs monoclonal antibodies
  • the upstream and recovery operations aims for high productivity of therapeutic protein in both cell culture and recovery process and various on-line configurations are available to monitor bioprocessing operations. See , Whitford W., Julien C. Bioprocess Int. (5), S32-S45 (2007). Recently real-time monitoring and controlling of cell culture process has been implemented. It has been shown that an increase in the non-viable sub- population in CHO cell culture can predict the onset of stationary phase, demonstrating the opportunity for a completely automated cell culture process as well as a reliable and reproducible control of fed-batch additions during culture expansion. Sitton G., Srienc F. J. Biotechnol. , 135 (2008), 174-180. Others have utilized multiple steps in the primary recovery process to remove biomass and clarify the feed stream for downstream column chromatography. Bink L.R., Furey J. BioProcess Int. 8(3) 2010, 44-49, 57 (2010).
  • the design of harvest skid has capabilities of real- time monitoring and controlling of critical process parameters and quality attributes.
  • the model disclosed herein can be applied to several processes with different cell properties and productivity level. With this system, start and end of clarified bulk collection can be determined in a quantitative way, which significantly improves harvest robustness and protein yield.
  • the methods disclosed herein provide a deep insight into the application of a harvest skid in cell culture clarification process.
  • the new harvest process disclosed herein improve protein yield while being scalable, auto-controllable, and applicable for multi- product with a wide range of properties.
  • the real-time titer information can be used to demonstrate cell culture performance and guide immediate downstream processing.
  • Disclosed herein is a method of monitoring in real-time a target protein concentration (titer) in a sample mixture comprising a target protein and impurities comprising monitoring in real-time an ultraviolet (UV) signal of the sample mixture and automatically transferring the UV signal into target protein titer using established models during a filtration based cell culture harvesting process.
  • UV ultraviolet
  • Also disclosed herein is a method of controlling target protein collection
  • the UV signal is continuously transferred to a titer of the target protein according to established models and automatic control.
  • the titer of the target protein is at least about 0.01 g/L, at least about 0.02 g/L, at least about 0.03 g/L, at least about 0.04 g/L, at least about 0.05 g/L, at least about 0.06 g/L, at least about 0.07 g/L, at least about 0.08 g/L, at least about 0.09 g/L, at least about 0.1 g/L, at least about 0.2 g/L, at least about 0.3 g/L, at least about 0.4 g/L, at least about 0.5 g/L, at least about 0.6 g/L, at least about 0.7 g/L, at least about 0.8 g/L, at least about 0.9 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 0.01 g/L
  • the methods disclosed herein further comprise beginning collection of the target protein when the titer is at least about 0.05 g/L, at least about 0.06 g/L, at least about 0.07 g/L, at least about 0.08 g/L, at least about 0.09 g/L, at least about 0.1 g/L, at least about 0.2 g/L, at least about 0.3 g/L, at least about 0.4 g/L, at least about 0.5 g/L, at least about 0.6 g/L, at least about 0.7 g/L, at least about 0.8 g/L, at least about 0.9 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, at least about 2.5 g/L, at least about 3 g/L, at least about 3.5 g/L, at least about 4 g/L, at least about 4.5 g/L, at least about 5 g/L, at least about
  • the titer that the target protein is collected is between about
  • 0.05 g/L and about 20 g/L between about 0.1 g/L and about 20 g/L, between about 0.2 g/L and about 20 g/L, between about 0.3 g/L and about 20 g/L, between about 0.4 g/L and about 20 g/L, between about 0.5 g/L and about 20 g/L, between about 0.6 g/L and about 20 g/L, between about 0.7 g/L and about 20 g/L, between about 0.8 g/L and about 20 g/L, between about 0.9 g/L and about 20 g/L, between about 1 g/L and about 20 g/L, between about 0.05 g/L and about 15 g/L, between about 0.1 g/L and about 15 g/L, between about 0.2 g/L and about 15 g/L, between about 0.3 g/L and about 15 g/L, between about 0.4 g/L and about 15 g/L, between about
  • the methods disclosed herein further comprise stopping the collection of the target protein when the collection titer is below about 0.1 or 0.2 g/L.
  • the target protein yield is increased at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% compared to the protein yield without monitoring in real time an ultraviolet (UV) signal of the sample mixture.
  • UV ultraviolet
  • the target protein is harvested from a culture medium having a cell density of at least about 1 X 10 6 cells/mL, at least about 5 X 10 6 cells/mL, at least about 1 X 10 7 cells/mL, at least about 1.5 X 10 7 cells/mL, at least about 2 X 10 7 cells/mL, at least about 2.5 X 10 7 cells/mL, at least about 3 X 10 7 cells/mL, at least about 3.5 X 10 7 cells/mL, at least about 4 X 10 7 cells/mL, at least about 4.5 X 10 7 cells/mL, or at least about 5 X 10 7 cells/mL.
  • the protein filtration is a depth filtration. In some embodiments, the protein filtration is a depth filtration.
  • the depth filtration comprises a primary depth filter and/or a secondary depth filter.
  • the methods disclosed herein further comprise loading the sample mixture prior to the monitoring. In some embodiments, the methods disclosed herein further comprise flushing the depth filters with water or buffer before loading the cell culture and chasing the depth filters post loading the cell culture. In some
  • the methods disclosed herein further comprise chasing the sample mixture with phosphate buffered saline (PBS) or other buffers.
  • the filtration based cell culture harvesting process comprises a harvest skid.
  • the harvest skid comprises a control system wherein the control system automatically starts collection of the protein when the set titer is achieved.
  • the harvest skid comprises a control system, wherein the control system automatically stops collection of the protein when the set titer is achieved.
  • the control system modulates flow rate of a liquid through the harvest skid.
  • the control system automatically drives the pump to up-regulate flow rate through the harvest skid.
  • the control system
  • the methods disclosed herein do not comprise a step of air blow- down.
  • the target protein titer or the protein yield is not based on volume.
  • a method of increasing, controlling, or modulating protein yield in a sample mixture comprising a target protein and impurities comprising (a) flushing a harvest skid with water; (b) loading the sample into the harvest skid; (c) measuring an ultraviolet (UV) signal of the sample mixture during protein filtration in the harvest skid into a real-time protein titer; (d) starting collection of the protein based on the UV measurement and the real-time protein titer; (e) chasing the protein with PBS; and (f) stopping collection of the protein based on the UV measurement and the real-time protein titer; wherein the UV signal correlates with the real-time protein titer during the filtration.
  • UV ultraviolet
  • the methods further comprise measuring pressure,
  • the methods further comprise measuring pressure using a pressure sensor.
  • the pressure is measured in a range of -10 pounds per square inch (psi) to 50 psi, -10 psi to 40 psi, -9 psi to 40 psi, -8 psi to 40 psi, -7 psi to 30 psi, -6 psi to -20 psi, -7 psi to 40 psi, -8 psi to 40 psi, -9 psi to 45 psi, -10 psi to -45 psi, or -7 psi to -45 psi.
  • the methods further comprise measuring turbidity.
  • the turbidity is measured in a range of 0 absorbance units (AU) to 2 AU.
  • the methods further comprise measuring temperature.
  • the temperature is measured in a range of 0°C to 70°C, 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 5°C to 70°C, l0°C to 70°C, l5°C to 70°C, 20°C to 70°C, l0°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, 20°C to 30°C, 35°C to 40°C, or 25°C to 45°C,.
  • the methods further comprise measuring flow.
  • the flow is measured in a range of 0 L/min to 20 L/min, .0 L/min to 30 L/min, 0 L/min to 40 L/min, 0 L/min to 50 L/min, 0 L/min to 60 L/min, 0 L/min to 70 L/min, 0 L/min to 80 L/min, 0 L/min to 90 L/min, 0 L/min to 100 L/min, 0 L/min to 110 L/min, 0 L/min to 120 L/min, 0 L/min to 130 L/min, 0 L/min to 140 L/min, 0 L/min to 150 L/min, 0 L/min to 160 L/min, 0 L/min to 170 L/min, 0 L/min to 180 L/min, 0 L/min to 190 L/min, 0 L/min to 200 L/min, 0 L/min to 250 L/min, or
  • the harvest skid comprises one or more filters.
  • the filters comprise a primary depth filter and a secondary depth filter.
  • the sample mixture is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any
  • the protein is produced in culture comprising mammalian cells.
  • the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK) or any combination thereof.
  • the protein comprises an antibody or a fusion protein.
  • the protein is an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti-LAG3 antibody and anti-IL8 antibody.
  • the protein is Abatacept or Belatacept.
  • a system for real time monitoring and controlling of protein yield comprising a sensor measuring a real-time UV signal of a sample mixture comprising a target protein and impurities.
  • the system further comprises a sensor measuring pressure, turbidity, temperature, flow, weight, or any combination thereof.
  • an apparatus comprises a sensor configured to measure a
  • a system comprises an apparatus comprising a sensor configured to measure a UV signal of a sample mixture comprising a target protein and impurities.
  • the system disclosed is for use in the methods described herein.
  • FIG. 1 A shows mechanical design of an exemplary harvest skid. All values are listed in inches.
  • FIG. 1B shows the physical picture of harvest skid.
  • FIG. 2 shows process flow chart for cell culture harvest process with new harvest skid.
  • Various boxes illustrate online measurement sensors, control modules and physical instruments.
  • FIG. 3 shows the experimental design described herein to model UV signal to product titer.
  • FIG. 4 shows a graphic comparison between old and new harvest methods.
  • the new method eliminates air blow-down step.
  • start and end of collections of clarified bulk in the new method can be controlled automatically based on online UV readings and calculated titer. More specifically, real-time target protein concentration during the harvest process can be calculated by online UV sensor readings, using the models generated and tested herein. Cut-off of bulk collection can therefore be determined directly on calculated online target protein concentration.
  • the calculation algorithms can be integrated into Delta VTM control system to achieve automatic cut-off of clarified bulk collection.
  • FIG. 5 shows offline titer measurements against online UV signals using serial dilution samples of GITR cell culture.
  • FIG. 6A and FIG. 6B show offline titer measurements against online UV signals with small scale harvest processes using pure protein (Fig. 6A) and clarified bulk (Fig. 6B). Online UV and offline titer values during the test harvest process were plotted.
  • FIG. 7A, FIG. 7B, and FIG. 7C show offline titer measurements against online
  • UV signals with large scale harvest processes using an anti-GITR antibody cell culture (Fig. 7A), Abatacept cell culture (Fig. 7B), and anti-CXCR4 antibody cell culture (Fig. 7C).
  • FIG. 8A and FIG. 8B show linear fit of offline titer measurements against online
  • FIG. 8A Linear fit of predicted titer based on UV against actual titer (FIG. 8B).
  • FIG. 9A and FIG. 9B show non-linear fit of offline titer measurements against online UV values (FIG. 9A); Linear fit of predicted titer based on UV against actual titer (FIG. 9B).
  • FIG. 10 shows mean difference between model -predicted titer and actual titer
  • FIG. 11 shows the comparison of online UV trace, titer trace achieved by
  • the Y axis shows titer (g/L) determined offline or titer modelled based on UV (g/L), and the X axis shows time (min).
  • the triangle line shows online UV
  • the square line shows titer modelled based on UV (g/L)
  • the diamond line shows offline titer (g/L).
  • Methods include controlling, modulating, or increasing protein yield using real-time measurement of an ultraviolet (UV) signal of a sample mixture during a purification step, e.g., protein filtration in a harvest skid.
  • UV ultraviolet
  • the methods utilize a UV signal to provide a titer of the target protein according to formulae disclosed herein, which differ based on whether collection occurs from the start of loading to the end of loading or after the end of loading.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • polypeptides are approximate, and are provided for description.
  • Modeling refers to the method of establishing a linear fit to determine the titer (e.g, in g/L) of a test protein.
  • modeling includes methods from start collection to end loading (e.g, incline modeling).
  • modeling includes start chasing to end collection (e.g, decline modeling).
  • modeling includes both the incline modeling and decline modeling.
  • Protein yield refers to the total amount of protein recovered after the processes disclosed herein. Protein yield can be measured in grams or in a final concentration (e.g, mg/ml) in a fixed volume. Percent yield can also be measures as a percentage of the amount of the starting protein (e.g, bulk enzyme).
  • controlling protein yield can refer to regulating, testing, or verifying the end product (e.g, a protein) collected during the processes disclosed herein. In some embodiments, controlling protein yield is achieved through altering the UV signal in real-time to affect critical process parameters and quality attributes and regulate protein yield. In some embodiments, controlling protein yield refers to maintaining a constant UV signal during the methods disclosed herein in order to achieve a desired protein yield.
  • modulating protein yield refers to changing, varying, or altering the end product (e.g a protein) collected during the processes disclosed herein. Modulating the protein yield alters the protein end-product yield, which can be increased, reduced, or inhibited. In some embodiments, the process modulates the protein yield, which results in an increase in protein yield. In some embodiments, modulating protein yield is achieved through altering the UV signal in real-time to affect critical process parameters and quality attributes and regulate protein yield.
  • a harvest skid as described herein comprising multiple sensors for real-time
  • a harvest skid comprises one or more pressure sensors, one or more flow sensors, one or more ultraviolet (UV) sensors, one or more weight sensors, one or more turbidity sensors, and/or one or more temperature sensors,
  • Titer refers to the amount, or the concentration, of a substance in a solution.
  • Titer is determined using both incline modeling and decline modeling as described herein.
  • the present disclosure is based on the capabilities of real-time UV monitoring and controlling of critical process parameters and quality attributes.
  • the present methods allow translation of online UV signal of clarified bulk to real-time titer of target product using modeling methods.
  • the present methods can then be used to automatically control harvest process and to improve process yield, robustness, and consistency.
  • the titer information can also be used to demonstrate cell culture performance and guide the immediate processing of downstream purification.
  • disclosed herein is a method of controlling or modulating protein yield in a sample mixture comprising a target protein and impurities comprising monitoring in real-time an ultraviolet (UV) signal of the sample mixture during protein filtration in a harvest skid.
  • UV ultraviolet
  • the disclosure includes a method of monitoring in real-time a target protein concentration (titer) in a sample mixture comprising a target protein and impurities comprising monitoring in real-time an ultraviolet (UV) signal of the sample mixture and automatically transferring the UV signal into target protein titer using established models during a filtration based cell culture harvesting process.
  • the disclosure provides a method of controlling target protein collection and improving protein yield in a sample mixture comprising a target protein and impurities comprising monitoring in real-time an ultraviolet (UV) signal of the sample mixture during a filtration based cell culture harvesting process.
  • Also disclosed herein is a method of increasing or improving protein yield in a sample mixture comprising a target protein and impurities comprising monitoring in real- time an ultraviolet (UV) signal of the sample mixture during a filtration based cell culture harvesting process, e.g., protein filtration in a harvest skid.
  • UV ultraviolet
  • Protein harvest/purification includes multiple steps to isolate or purify a target protein from the mixture of the protein with impurities, such as cells, cell culture medium, DNA, RNA, other proteins, etc.
  • Clarifying cell culture broth can be the first downstream unit operation in an elaborate sequence of steps required to purify a target protein.
  • a combination of centrifugation and/or filtration, e.g, depth filtration, is used for that operation.
  • the availability of large scale, filtration technology, e.g, depth filtration, that can monitor real-time protein concentration can thus provide the capability to improve and simplify downstream processes.
  • the depth filtration system can utilize a harvest skid as shown in FIG. 2. Before harvest, depth filters are flushed with water or an appropriate buffer to remove loose particulates and extractables from the filter manufacturing process.
  • the harvest skid can comprise one filter or multiple filters, e.g, a primary depth filter and a second depth filter.
  • Cell culture medium including a target protein can be obtained from a bioreactor and can be loaded onto (or pumped onto) one or more filters, e.g, a primary filter and a secondary filter.
  • the real-time UV signal can be then measured after the loaded cell culture medium has passed through the filter system, e.g, primary filter or the secondary filter.
  • the filtered product can then be obtained at one or more tank. Once a harvest is completed, the filters are again flushed to recover valuable product held-up in the housings. Harvest yields between 50% and 90% can be achievable using a post-use flush and ensuring minimum product loss.
  • the present methods are thus intended to improve the protein harvest yields at least by 1%, at least by 2%, at least by 3%, at least by 4%, at least by 5%, at least by 6%, at least by 7%, at least by 8%, at least by 9%, at least by 10%, at least by 11%, at least by 12%, at least by 13%, at least by 14%, at least by 15%, at least by 16%, at least by 17%, at least by 18%, at least by 19%, at least by 20%, at least by 21%, at least by 22%, at least by 23%, at least by 24%, or at least by 25%.
  • the UV signal provides a titer of the target protein from the start of the loading to the end of the loading and/or after the end of the loading till the end of filtration.
  • the titer of the target proteins from the start of the loading to the end of the loading can be calculated according to formula (I):
  • the titer of the target proteins from the start of the loading to the end of the loading can be calculated according to formula (I), which comprises constants (a) and (b).
  • (a) is a value between 0 and -1.0. In some embodiments,
  • (a) is a value between -0.1 and -0.9. In some embodiments, (a) is a value between -0.2 and -0.8. In some embodiments, (a) is a value between -0.3 and -0.7. In some
  • (a) is a value between -0.4 and -0.6.
  • (a) is a value between -0.2 and -0.5. In some embodiments,
  • (a) is a value between -0.25 and -0.45. In some embodiments, (a) is a value between -0.30 and -0.40.
  • (a) is a value between -0.5 and -0.9. In some embodiments,
  • (a) is a value between -0.55 and -0.85. In some embodiments, (a) is a value between -0.60 and -0.80. In some embodiments, (a) is a value between -0.65 and -0.75.
  • (a) is about -0.1. In some embodiments, (a) is about -0.15.
  • (a) is about -0.2. In some embodiments, (a) is about -0.25. In some embodiments, (a) is about -0.3. In some embodiments, (a) is about -0.35. In some embodiments, (a) is about -0.4. In some embodiments, (a) is about -0.45. In some embodiments, (a) is about -0.5. In some embodiments, (a) is about -0.55. In some embodiments, (a) is about -0.6. In some embodiments, (a) is about -0.65. In some embodiments, (a) is about -0.7. In some embodiments, (a) is about -0.75. In some embodiments, (a) is about -0.8. In some embodiments, (a) is about -0.85. In some embodiments, (a) is about -0.9. In some embodiments, (a) is about -0.95. In some embodiments, (a) is about -1.0.
  • (a) is -0.35. In some embodiments, (a) is -0.69. In one embodiment, the cell type is DG44 and (a) is -0.35. In one embodiment, the cell type is CHOZN, and (a) is -0.69. [0073] In some embodiments, (b) is a value between 1.0 and 5.0. In some embodiments,
  • (b) is a value between 1.5 and 4.5. In some embodiments, (b) is a value between 2.0 and 4.0. In some embodiments, (b) is a value between 2.5 and 3.5.
  • (b) is a value between 2.0 and 3.6. In some embodiments,
  • (b) is a value between 2.1 and 3.5. In some embodiments, (b) is a value between 2.2 and 3.4. In some embodiments, (b) is a value between 2.3 and 3.3. In some embodiments, (b) is a value between 2.4 and 3.2. In some embodiments, (b) is a value between 2.5 and 3.1. In some embodiments, (b) is a value between 2.6 and 3.0. In some embodiments, (b) is a value between 2.7 and 2.9.
  • (b) is a value between 3.3 and 4.8. In some embodiments,
  • (b) is a value between 3.4 and 4.7. In some embodiments, (b) is a value between 3.5 and 4.6. In some embodiments, (b) is a value between 3.6 and 4.5. In some embodiments, (b) is a value between 3.7 and 4.4. In some embodiments, (b) is a value between 3.8 and 4.3. In some embodiments, (b) is a value between 3.9 and 4.2. In some embodiments, (b) is a value between 4.0 and 4.1.
  • (b) is about 2.0. In some embodiments, (b) is about 2.1. In some embodiments, (b) is about 2.2. In some embodiments, (b) is about 2.3. In some embodiments, (b) is about 2.4. In some embodiments, (b) is about 2.5. In some embodiments, (b) is about 2.6. In some embodiments, (b) is about 2.7. In some embodiments, (b) is about 2.8. In some embodiments, (b) is about 2.9. In some embodiments, (b) is about 3.0. In some embodiments, (b) is about 3.1. In some embodiments, (b) is about 3.2. In some embodiments, (b) is about 3.3.
  • (b) is about 3.4. In some embodiments, (b) is about 3.5. In some embodiments, (b) is about 3.6. In some embodiments, (b) is about 3.7. In some embodiments, (b) is about 3.8. In some embodiments, (b) is about 3.9. In some embodiments, (b) is about 4.0. In some embodiments, (b) is about 4.1. In some embodiments, (b) is about 4.2. In some embodiments, (b) is about 4.3. In some embodiments, (b) is about 4.4. In some embodiments, (b) is about 4.5. In some embodiments, (b) is about 4.6. In some embodiments, (b) is about 4.7. In some embodiments, (b) is about 4.8. In some embodiments, (b) is about 4.9. In some embodiments, (b) is about 5.0.
  • (b) is 2.88. In some embodiments, (b) is 4.06. In one
  • the cell type is DG44 and (b) is 2.88. In one embodiment, the cell type is CHOZN, and (b) is 4.06. In some embodiments, (a) is -0.35 and (b) is 2.88. In some embodiments, (a) is -0.69 and (b) is 4.06. In one embodiment, the cell type is DG44, and (a) is -0.35 and (b) is 2.88. In one embodiment, the cell type is CHOZN, and (a) is -0.69 and (b) is 4.06.
  • the titer of the target proteins after the end of the loading till the end of filtration can be calculated according to formula (II):
  • the titer of the target proteins from the start of the loading to the end of the loading can be calculated according to formula (II), which comprises constants (A), (B), and (C).
  • (A) is a value between -2.5 and 1.0. In some embodiments,
  • (A) is a value between -2.0 and 0.5. In some embodiments, (A) is a value between -1.5 and 0.0. In some embodiments, (A) is a value between -1.0 and -0.5.
  • (A) is a value between -1.5 and -0.4. In some embodiments,
  • (A) is a value between -1.4 and -0.5. In some embodiments, (A) is a value between -1.3 and -0.6. In some embodiments, (A) is a value between -1.2 and -0.7. In some embodiments, (A) is a value between -1.1 and -0.8. In some embodiments, (A) is a value between -1.0 and -0.9.
  • (A) is a value between -1.0 and 1.0. In some embodiments,
  • (A) is a value between -0.9 and 0.9. In some embodiments, (A) is a value between -0.8 and 0.8. In some embodiments, (A) is a value between -0.7 and 0.7. In some
  • (A) is a value between -0.6 and 0.6. In some embodiments, (A) is a value between -0.5 and 0.5. In some embodiments, (A) is a value between -0.4 and 0.4. In some embodiments, (A) is a value between -0.3 and 0.3. In some embodiments, (A) is a value between -0.2 and 0.2. In some embodiments, (A) is a value between -0.1 and 0.1.
  • (A) is about -2.0. In some embodiments, (A) is about -1.9.
  • (A) is about -1.8. In some embodiments, (A) is about -1.7. In some embodiments, (A) is about -1.6. In some embodiments, (A) is about -1.5. In some embodiments, (A) is about -1.4. In some embodiments, (A) is about -1.3. In some embodiments, (A) is about -1.2. In some embodiments, (A) is about -1.1. In some embodiments, (A) is about -1.0. In some embodiments, (A) is about -0.9. In some embodiments, (A) is about -0.8. In some embodiments, (A) is about -0.7. In some embodiments, (A) is about -0.6.
  • (A) is about -0.5. In some embodiments, (A) is about -0.4. In some embodiments, (A) is about -0.3. In some embodiments, (A) is about -0.2. In some embodiments, (A) is about -0.1. In some embodiments, (A) is about 0.1. In some embodiments, (A) is about 0.2. In some embodiments, (A) is about 0.3. In some embodiments, (A) is about 0.4. In some embodiments, (A) is about 0.5. In some embodiments, (A) is about 0.6. In some embodiments, (A) is about 0.7. In some embodiments, (A) is about 0.8. In some embodiments, (A) is about 0.9. In some embodiments, (A) is about 1.0.
  • (A) is -0.95. In some embodiments, (A) is 0.02. In one embodiment, the cell type is DG44 and (A) is -0.95. In one embodiment, the cell type is CHOZN and (A) is 0.02.
  • (B) is a value between -1.5 and 2.5. In some embodiments,
  • (B) is a value between -1.0 and 2.0. In some embodiments, (B) is a value between -0.5 and 1.5. In some embodiments, (B) is a value between 0 and 1.0.
  • (B) is a value between -0.5 and -0.4. In some embodiments,
  • (B) is a value between -0.4 and -0.3. In some embodiments, (B) is a value between -0.3 and -0.2. In some embodiments, (B) is a value between -0.2 and -0.1. In some embodiments, (B) is a value between -0.1 and 0.0. In some embodiments, (B) is a value between 0.0 and 0.1. In some embodiments, (B) is a value between 0.1 and 0.2. In some embodiments, (B) is a value between 0.2 and 0.3. In some embodiments, (B) is a value between 0.3 and 0.4. In some embodiments, (B) is a value between 0.4 and 0.5.
  • (B) is a value between 0.5 and 0.6. In some embodiments, (B) is a value between 0.6 and 0.7. In some embodiments, (B) is a value between 0.7 and 0.8. In some embodiments, (B) is a value between 0.8 and 0.9. In some embodiments, (B) is a value between 0.9 and 1.0. In some embodiments, (B) is a value between 1.0 and 1.1. In some embodiments, (B) is a value between 1.1 and 1.2. In some embodiments, (B) is a value between 1.2 and 1.3. In some embodiments, (B) is a value between 1.3 and 1.4. In some embodiments, (B) is a value between 1.4 and 1.5.
  • (B) is about -1.5. In some embodiments, (B) is about -1.4.
  • (B) is about -1.3. In some embodiments, (B) is about -1.2. In some embodiments, (B) is about -1.1. In some embodiments, (B) is about -1.0. In some embodiments, (B) is about -0.9. In some embodiments, (B) is about -0.8. In some embodiments, (B) is about -0.7. In some embodiments, (B) is about -0.6. In some embodiments, (B) is about -0.5. In some embodiments, (B) is about -0.4. In some embodiments, (B) is about -0.3. In some embodiments, (B) is about -0.2. In some embodiments, (B) is about -0.1.
  • (B) is about 0.1. In some embodiments, (B) is about 0.2. In some embodiments, (B) is about 0.3. In some embodiments, (B) is about 0.4. In some embodiments, (B) is about 0.5. In some embodiments, (B) is about 0.6. In some embodiments, (B) is about 0.7. In some embodiments, (B) is about 0.8. In some embodiments, (B) is about 0.9. In some embodiments, (B) is about 1.0. In some embodiments, (B) is about 1.1. In some embodiments, (B) is about 1.2. In some embodiments, (B) is about 1.3. In some embodiments, (B) is about 1.4.
  • (B) is about 1.5. In some embodiments, (B) is about 1.6. In some embodiments, (B) is about 1.7. In some embodiments, (B) is about 1.8. In some embodiments, (B) is about 1.9. In some embodiments, (B) is about 2.0.
  • (B) is 0.86. In some embodiments, (B) is 0.13. In one embodiment, the cell type is DG44 and (B) is 0.86. In one embodiment, the cell type is CHOZN and (B) is 0.13.
  • (C) is a value between 0 and 4.0. In some embodiments, (C) is a value between 0.5 and 3.5. In some embodiments, (C) is a value between 1.0 and 3.0. In some embodiments, (C) is a value between 1.5 and 2.5.
  • (C) is a value between 0.0 and 0.1. In some embodiments, (C) is a value between 0.1 and 0.2. In some embodiments, (C) is a value between 0.2 and 0.3. In some embodiments, (C) is a value between 0.3 and 0.4. In some embodiments, (C) is a value between 0.4 and 0.5. In some embodiments, (C) is a value between 0.5 and 0.6. In some embodiments, (C) is a value between 0.6 and 0.7. In some embodiments, (C) is a value between 0.7 and 0.8. In some embodiments, (C) is a value between 0.8 and 0.9.
  • (C) is a value between 0.9 and 1.0. In some embodiments, (C) is a value between 1.0 and 1.1. In some embodiments, (C) is a value between 1.1 and 1.2. In some embodiments, (C) is a value between 1.2 and 1.3. In some embodiments, (C) is a value between 1.3 and 1.4. In some embodiments, (C) is a value between 1.4 and 1.5. In some embodiments, (C) is a value between 1.5 and 1.6. In some embodiments, (C) is a value between 1.6 and 1.7. In some embodiments, (C) is a value between 1.7 and 1.8. In some embodiments, (C) is a value between 1.8 and 1.9.
  • (C) is a value between 1.9 and 2.0. In some embodiments, (C) is a value between 2.0 and 2.1. In some embodiments, (C) is a value between 2.1 and 2.2. In some embodiments, (C) is a value between 2.2 and 2.3. In some embodiments, (C) is a value between 2.3 and 2.4. In some embodiments, (C) is a value between 2.4 and 2.5. In some embodiments, (C) is a value between 2.5 and 2.6. In some embodiments, (C) is a value between 2.6 and 2.7. In some embodiments, (C) is a value between 2.7 and 2.8. In some embodiments, (C) is a value between 2.8 and 2.9.
  • (C) is a value between 2.9 and 3.0. In some embodiments, (C) is a value between 3.0 and 3.1. In some embodiments, (C) is a value between 3.1 and 3.2. In some embodiments, (C) is a value between 3.2 and 3.3. In some embodiments, (C) is a value between 3.3 and 3.4. In some embodiments, (C) is a value between 3.4 and 3.5. In some embodiments, (C) is a value between 3.5 and 3.6. In some embodiments, (C) is a value between 3.6 and 3.7. In some embodiments, (C) is a value between 3.7 and 3.8. In some embodiments, (C) is a value between 3.8 and 3.9. In some embodiments, (C) is a value between 3.9 and 4.0.
  • (C) is 1.21. In some embodiments, (C) is 2.41. In one embodiment, the cell type is DG44 and (C) is 1.21. In one embodiment, the cell type is CHOZN and (C) is 2.41.
  • the cell type is DG44 and (A) is - 0.95, (B) is 0.86, and (C) is 1.21. In one embodiment, the cell type is CHOZN, and (A) is 0.02, (B) is 0.13, and (C) is 2.41.
  • a method of increasing, controlling, or modulating protein yield in a sample mixture comprising a target protein and impurities comprising (a) flushing a harvest skid with water; (b) loading the sample into the harvest skid; (c) measuring an ultraviolet (UV) signal of the sample mixture during protein filtration in the harvest skid into a real-time protein titer; (d) starting collection of the protein based on the UV measurement and the real-time protein titer; (e) chasing the protein with PBS; and (f) stopping collection of the protein based on the UV
  • UV ultraviolet
  • UV signal correlates with the real-time protein titer during the filtration.
  • the methods described herein comprise a water (e.g.
  • the methods comprise loading the protein sample and starting collection based on an online titer. In some embodiments, the methods comprise a PBS chase and an end collection based on an online titer. Compared with other methods, the methods disclosed herein do not comprise an air blow-down step.
  • the start and end of collection of sample are controlled automatically based on online UV readings and calculated titer.
  • the real-time target protein concentration during the harvest process is calculated by online UV sensor readings, using modeling.
  • cut-off of bulk collection is determined directly on calculated online target protein concentration.
  • the calculation algorithms are integrated into Delta VTM control system to achieve automatic cut-off of protein collection.
  • the methods disclosed herein comprise a step of modeling.
  • modeling comprises offline titer measurements against online UV signals using serial dilution samples to establish a linear correlation between UV signal and titer.
  • the sample used in modeling is a purified protein.
  • the sample used in modeling is a bulk protein that comprises contaminants.
  • the modeling is then used to control, modulate, increase, and/or improve protein yield.
  • the methods disclosed herein comprise controlling,
  • the titer is at least about 0.01 g/L.
  • the titer is at least about 0.02 g/L.
  • the titer is at least about 0.03 g/L.
  • the titer is at least about 0.04 g/L.
  • the titer is at least about 0.05 g/L.
  • the titer is at least about 0.06 g/L.
  • the titer is at least about 0.07 g/L.
  • the titer is at least about 0.08 g/L.
  • the titer is at least about 0.09 g/L. In some embodiments, the titer is at least about 0.1 g/L. In some embodiments, the titer is at least about 0.2 g/L. In some embodiments, the titer is at least about 0.3 g/L. In some embodiments, the titer is at least about 0.4 g/L. In some embodiments, the titer is at least about 0.5 g/L. In some embodiments, the titer is at least about 0.6 g/L. In some embodiments, the titer is at least about 0.7 g/L. In some embodiments, the titer is at least about 0.8 g/L. In some embodiments, the titer is at least about 0.9 g/L. In some embodiments, the titer is at least about 1 g/L. In some embodiments, the titer is at least about 0.09 g/L. In some embodiments, the t
  • the titer is at least about 1.5 g/L. In some embodiments, the titer is at least about 2 g/L. In some embodiments, the titer is at least about 2.5 g/L. In some
  • the titer is at least about 3 g/L. In some embodiments, the titer is at least about 3.5 g/L. In some embodiments, the titer is at least about 4 g/L. In some
  • the titer is at least about 4.5 g/L. In some embodiments, the titer is at least about 5 g/L. In some embodiments, the titer is at least about 5.5 g/L. In some
  • the titer is at least about 6 g/L. In some embodiments, the titer is at least about 6.5 g/L. In some embodiments, the titer is at least about 7 g/L. In some
  • the titer is at least about 7.5 g/L. In some embodiments, the titer is at least about 8 g/L. In some embodiments, the titer is at least about 8.5 g/L. In some embodiments, the titer is at least about 9 g/L. In some embodiments, the titer is at least about 9.5 g/L. In some embodiments, the titer is at least about 10 g/L. In some embodiments, the titer is at least about 10.5 g/L. In some embodiments, the titer is at least about 11 g/L. In some embodiments, the titer is at least about 11.5 g/L.
  • the titer is at least about 12 g/L. In some embodiments, the titer is at least about 12.5 g/L. In some embodiments, the titer is at least about 13 g/L. In some embodiments, the titer is at least about 13.5 g/L. In some embodiments, the titer is at least about 14 g/L. In some embodiments, the titer is at least about 14.5 g/L. In some embodiments, the titer is at least about 15 g/L. In some embodiments, the titer is at least about 15.5 g/L. In some embodiments, the titer is at least about 16 g/L.
  • the titer is at least about 16.5 g/L. In some embodiments, the titer is at least about 17 g/L. In some embodiments, the titer is at least about 17.5 g/L. In some embodiments, the titer is at least about 18 g/L, at least about 18.5 g/L. In some embodiments, the titer is at least about 19 g/L. In some embodiments, the titer is at least about 19.5 g/L. In some embodiments, the titer is at least about 20 g/L.
  • the methods disclosed herein comprise collection of the target protein that is dependent on the titer of the target protein.
  • collection of the target protein begins when the titer is at least about 0.05 g/L.
  • collection of the target protein begins when the titer is at least about 0.06 g/L.
  • collection of the target protein begins when the titer is at least about 0.07 g/L.
  • collection of the target protein begins when the titer is at least about 0.08 g/L.
  • collection of the target protein begins when the titer is at least about 0.09 g/L.
  • collection of the target protein begins when the titer is at least about 0.1 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.2 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.3 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.4 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.6 g/L.
  • collection of the target protein begins when the titer is at least about 0.7 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.8 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 0.9 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 1 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 1.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 2 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 2.5 g/L.
  • collection of the target protein begins when the titer is at least about 3 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 3.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 4 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 4.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 5.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 6 g/L.
  • collection of the target protein begins when the titer is at least about 6.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 7 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 7.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 8 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 8.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 9 g/L.
  • collection of the target protein begins when the titer is at least about 9.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 10 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 10.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 11 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 11.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 12 g/L.
  • collection of the target protein begins when the titer is at least about 12.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 13 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 13.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 14 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 14.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 15 g/L.
  • collection of the target protein begins when the titer is at least about 15.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 16 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 16.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 17 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 17.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 18 g/L.
  • collection of the target protein begins when the titer is at least about 18.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 19 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 19.5 g/L. In some embodiments, collection of the target protein begins when the titer is at least about 20 g/L.
  • the methods disclosed herein comprise collection of a target protein, wherein the target protein has a titer in a range.
  • the titer at which the target protein is collected is between about 0.05 g/L and about 20 g/L.
  • the titer at which the target protein is collected is between about 0.1 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.2 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.3 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.4 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is, between about 0.5 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.6 g/L and about 20 g/L. In some
  • the titer at which the target protein is collected is between about 0.7 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.8 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.9 g/L and about 20 g/L. In some
  • the titer at which the target protein is collected is between about 1 g/L and about 20 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.05 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.1 g/L and about 15 g/L. In some
  • the titer at which the target protein is collected is between about 0.2 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.3 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.4 g/L and about 15 g/L. In some
  • the titer at which the target protein is collected is between about 0.5 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.6 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.7 g/L and about 15 g/L. In some
  • the titer at which the target protein is collected is between about 0.8 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.9 g/L and about 15 g/L. In some embodiments, the titer at which the target protein is collected is between about 1 g/L and about 15 g/L. In some
  • the titer at which the target protein is collected is between about 0.05 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.1 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.2 g/L and about 10 g/L. In some
  • the titer at which the target protein is collected is between about 0.3 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.4 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.5 g/L and about 10 g/L. In some
  • the titer at which the target protein is collected is between about 0.6 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.7 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 0.8 g/L and about 10 g/L. In some
  • the titer at which the target protein is collected is between about 0.9 g/L and about 10 g/L. In some embodiments, the titer at which the target protein is collected is between about 1 g/L and about 10 g/L.
  • the methods disclosed herein further comprise stopping the collection of the target protein when the collection titer is below about 0.5 g/L.
  • the yield of the target protein is increased by the methods disclosed herein. In some embodiments, the target protein yield is increased at least about 1% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 2% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 3% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 4% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture.
  • UV ultraviolet
  • the target protein yield is increased at least about 5% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 6% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 7% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 8% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture.
  • UV ultraviolet
  • the target protein yield is increased at least about 9% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 10% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 11% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 12% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture.
  • the target protein yield is increased at least about 13% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 14% compared to the protein yield without monitoring in real- time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 15% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 16% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture.
  • the target protein yield is increased at least about 17% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 18% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased at least about 19% compared to the protein yield without monitoring in real- time an ultraviolet (UV) signal of the sample mixture. In some embodiments, the target protein yield is increased or at least about 20% compared to the protein yield without monitoring in real-time an ultraviolet (UV) signal of the sample mixture.
  • UV ultraviolet
  • the ultraviolet (UV) signal of the sample mixture is
  • the UV signal of the sample mixture is measured at about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9 AU, about 1.0 AU, about 1.1 AU, about 1.2 AU, about 1.3 AU, about 1.4 AU, about 1.5 AU, about 1.6 AU, about 1.7 AU, about 1.8 AU, about 1.9 AU, or about 2.0 AU.
  • the methods comprise protein filtration.
  • the methods comprises one or more filters.
  • the protein filtration is a depth filtration.
  • the depth filtration comprises a primary depth filter and a secondary depth filter.
  • the depth filtration comprises a primary depth filter.
  • the methods comprise loading the sample mixture prior to the monitoring.
  • the methods comprise flushing the depth filters with a buffer before loading the cell culture and chasing the depth filters post loading the cell culture.
  • the methods comprise chasing the sample mixture with phosphate buffered saline (PBS).
  • the methods comprise a harvest skid which comprises a control system wherein the control system automatically starts collection of the protein when the titer is above 0.5 g/L.
  • the methods comprise a harvest skid comprises a control system, wherein the control system automatically stops collection of the protein when the titer is below 0.5 g/L.
  • the methods comprise a control system modulates flow rate of a liquid through the harvest skid. In some embodiments, the methods comprise a control system that automatically drives the pump to up-regulate flow rate through the harvest skid. In some embodiments, the methods comprise a control system that automatically drives the pump to down-regulate flow rate through the harvest skid. In some embodiments, the methods do not comprise a step of air blow-down.
  • the methods comprise a step of collecting a protein yield that is not based on volume.
  • the methods disclosed herein comprise measuring pressure, turbidity, temperature, flow rate, or any combination thereof.
  • the methods comprise measuring pressure using a pressure sensor.
  • the pressure is measured in a range of -10 pounds per square inch (psi) to 50 psi, -10 psi to 40 psi, -9 psi to 40 psi, -8 psi to 40 psi, -7 psi to 30 psi, -6 psi to -20 psi, -7 psi to 40 psi, -8 psi to 40 psi, -9 psi to 45 psi, -10 psi to -45 psi, or -7 psi to -45 psi.
  • the pressure can be measured at least once, twice, three times, four times, or five times, e.g., before the
  • the methods comprise measuring turbidity. In some embodiments, the methods comprise measuring turbidity.
  • the turbidity is measured in a range of 0 absorbance units (AU) to 2 AU. In other embodiments, the turbidity is measured at about 0.1 AU, about 0.2 AU, about 0.3 AU, about 0.4 AU, about 0.5 AU, about 0.6 AU, about 0.7 AU, about 0.8 AU, about 0.9
  • the turbidity is measured at least once, twice, three times, four times, or five times, e.g, after the primary filter, after the secondary filter, or after the primary filter and the secondary filter. See Fig. 2.
  • the methods comprise measuring temperature.
  • the temperature is measured in a range of 0°C to 70°C, 0°C to 60°C, 0°C to 50°C, 0°C to 40°C, 5°C to 70°C, l0°C to 70°C, l5°C to 70°C, 20°C to 70°C, l0°C to 60°C, 20°C to 50°C, 20°C to 40°C, 20°C to 45°C, 30°C to 40°C, 35°C to 40°C, 20°C to 30°C, 35°C to 40°C, or 25°C to 45°C.
  • the temperature can be measured any time during the filtration process, e.g, at least once, twice, three times, four times, or five times, e.g, after the primary filter, after the secondary filter, or after the primary and the secondary filters. See Fig. 2.
  • the flow is measured in a range of 0 L/min to 20 L/min, .0 L/min to 30 L/min, 0 L/min to 40 L/min, 0 L/min to 50 L/min, 0 L/min to 60 L/min, 0 L/min to 70 L/min, 0 L/min to 80 L/min, 0 L/min to 90 L/min, 0 L/min to 100 L/min, 0 L/min to 110 L/min, 0 L/min to 120 L/min, 0 L/min to 130 L/min, 0 L/min to 140 L/min, 0 L/min to 150 L/min, 0 L/min to 160 L/min, 0 L/min to 170 L/min, 0 L/min to 180 L/min, 0 L/min to 190 L/min, 0 L/min to 200 L/min, 0 L/min to 250 L/min, or 0 L/min to 300 L/min. In other embodiments, the flow is
  • liquid from water source/bioreactor/PBS source is driven to primary depth filter by the LEVITRONIX® gravity pump.
  • a system e.g., Delta VTM
  • flow totalizer volume is used to determine the end of water flush.
  • four pressure sensors are placed before primary depth filter, secondary depth filter, pre-filter and sterile filter. Pressure-flow control loop can work based on real-time pressure value before the primary depth filter. If the pressure values exceed a certain threshold, Delta VTM automatically drives the pump to down-regulate flow rate.
  • two turbidity sensors are placed after primary and secondary depth filters as indicators of filtrate quality.
  • one UV sensor value of which is used to calculate online target protein concentration and control cut-off of clarified bulk collection, is placed after the secondary depth filter.
  • Real-time upstream source and downstream receiving vessel weights were monitored and displayed on Delta VTM as well.
  • weight is monitors from 0 to 550 kg, with a measurement accuracy of 0.01 kg.
  • protein is isolated from a source.
  • the sample mixture is selected from the group consisting of a pure protein sample, a clarified bulk protein sample, a cell culture sample, and any combination thereof.
  • the source is selected from cultured cells.
  • the cells are prokaryotes.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the protein molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of a protein molecule, vectors which direct the expression of high levels of protein products that are readily purified can be desirable.
  • the cells are eukaryotes.
  • the cells are mammalian cells.
  • the cells are selected from Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK), and any combination thereof.
  • the cells are Chinese hamster ovary cells.
  • the cells are insect cells, e.g., Spodoptera frugiperda cells.
  • the cells are mammalian cells.
  • mammalian cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO, CRL7030, COS (e.g, COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.
  • the mammalian cells are CHO cells.
  • the CHO cell is CHO-DG44, CHOZN, CHO/dhfr-, CHOK1SV GS-KO, or CHO-S.
  • the CHO cell is CHO-DG4.
  • the CHO cell is CHOZN.
  • CHO-K e.g., CHO Kl
  • the target protein is harvested from a culture medium
  • the target protein is harvested from a culture medium having a cell density of at least about 5xl0 6 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about lxlO 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about l.5xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 2xl0 7 cells/mL.
  • the target protein is harvested from a culture medium having a cell density of at least about 2.5xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 3xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 3.5xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 4xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 4.5xl0 7 cells/mL. In some embodiments, the target protein is harvested from a culture medium having a cell density of at least about 5xl0 7 cells/mL.
  • the source of the protein is bulk protein. In some embodiments, the source of the protein is bulk protein.
  • the source of the protein is a composition comprising protein and non- protein components.
  • the non-protein components can include DNA and other contaminant.
  • the source of the protein is from an animal. In some embodiments the source of the protein is from an animal.
  • the animal is a mammal such as a non-primate (e.g ., cow, pig, horse, cat, dog, rat etc.) or a primate (e.g., monkey or human).
  • the source is a tissue or cells from a human.
  • such terms refer to a non-human animal (e.g, a non-human animal such as a pig, horse, cow, cat or dog).
  • such terms refer to a pet or farm animal.
  • such terms refer to a human.
  • the proteins purified by the methods described herein are fusion proteins.
  • a "fusion" or “fusion” protein comprises a first amino acid sequence linked in frame to a second amino acid sequence with which it is not naturally linked in nature.
  • the amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide.
  • a fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • a fusion protein can further comprise a second amino acid sequence associated with the first amino acid sequence by a covalent, non-peptide bond or a non- covalent bond.
  • a single protein is made.
  • multiple proteins, or fragments thereof can be incorporated into a single polypeptide.
  • "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between two polypeptides fuses both polypeptides together in frame to produce a single polypeptide fusion protein.
  • the fusion protein further comprises a third polypeptide which, as discussed in further detail below, can comprise a linker sequence.
  • the proteins purified by the methods described herein are antibodies.
  • Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’) 2 fragments, disulfide- linked Fvs (sdFv), anti-idiotypic (anti-id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above.
  • antibodies described herein refer to polyclonal antibody populations.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi or IgA 2 ), or any subclass (e.g, IgG 2a or IgG 2b ) of immunoglobulin molecule.
  • antibodies described herein are IgG antibodies, or a class (e.g, human IgGi or IgG 4 ) or subclass thereof.
  • the antibody is a humanized monoclonal antibody.
  • the antibody is a human monoclonal antibody, preferably that is an immunoglobulin.
  • an antibody described herein is an IgGi, or IgG antibody.
  • the protein described herein is an“antigen-binding
  • antigen-binding region refers to a portion of an antibody molecule which comprises the amino acid residues that confer on the antibody molecule its specificity for the antigen (e.g, the complementarity determining regions (CDR)).
  • the antigen-binding region can be derived from any animal species, such as rodents (e.g, mouse, rat or hamster) and humans.
  • the protein is an anti-LAG3 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-NKG2a antibody, an anti-ICOS antibody, an anti-CDl37 antibody, an anti-KIR antibody, an anti-TGFp antibody, an anti-IL-lO antibody, an anti-B7-H4 antibody, an anti-Fas ligand antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-GITR antibody, an anti-CXCR4 antibody, an anti-CD73 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, an anti -PD- 1 antibody, an anti-PD-Ll antibody, an anti-IL8 antibody, or any combination thereof.
  • the protein is Abatacept NGP. In other embodiments, the protein is Belatacept NGP.
  • the protein is an anti-GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) antibody.
  • the anti- GITR antibody has the CDR sequences of 6C8, e.g, a humanized antibody having the CDRs of 6C8, as described, e.g., in W02006/105021; an antibody comprising the CDRs of an anti-GITR antibody described in WO2011/028683; an antibody comprising the CDRs of an anti-GITR antibody described in JP2008278814, an antibody comprising the CDRs of an anti- GITR antibody described in WO2015/031667, WO2015/187835, WO2015/184099, WO2016/054638, WO2016/057841, WO2016/057846, WO
  • the protein is an anti-LAG3 antibody.
  • Lymphocyte- activation gene 3 also known as LAG-3, is a protein which in humans is encoded by the LAG3 gene.
  • LAG3 which was discovered in 1990 and is a cell surface molecule with diverse biologic effects on T cell function. It is an immune checkpoint receptor and as such is the target of various drug development programs by pharmaceutical companies seeking to develop new treatments for cancer and autoimmune disorders. In soluble form it is also being developed as a cancer drug in its own right.
  • anti-LAG3 antibodies include, but are not limited to, the antibodies in WO 2017/087901 A2, WO 2016/028672 Al, WO 2017/106129 Al, WO 2017/198741 Al, US 2017/0097333 Al,
  • the protein is an anti-CXCR4 antibody.
  • CXCR4 is a 7 transmembrane protein, coupled to Gl.
  • CXCR4 is widely expressed on cells of hemopoietic origin, and is a major co-receptor with CD4+ for human immunodeficiency virus 1 (HIV-l) See Feng, Y., Broeder, C.C., Kennedy, P. E., and Berger, E. A. (1996) Science 272, 872-877.
  • anti-CXCR4 antibodies include, but are not limited to, the antibodies in WO 2009/140124 Al, US 2014/0286936 Al, WO 2010/125162 Al, WO 2012/047339 A2, WO 2013/013025 A2, WO 2015/069874 Al, WO 2008/142303 A2, WO 2011/121040 Al, WO 2011/154580 Al, WO 2013/071068 A2, and WO
  • the protein is an anti-CD73 (ecto-5 '-nucleotidase)
  • the anti-CD73 antibody inhibits the formation of adenosine. Degradation of AMP into adenosine results in the generation of an
  • anti-CD73 antibodies include, but are not limited to, the antibodies in WO 2017/100670 Al, WO 2018/013611 Al, WO 2017/152085 Al, and WO 2016/075176 Al, all of which are incorporated herein in their entireties.
  • the protein is an anti-TIGIT (T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunoreceptor with T cell Immunor
  • TIGIT is a member of the PVR (poliovirus receptor) family of immunoglobin proteins. TIGIT is expressed on several classes of T cells including follicular B helper T cells (TFH). The protein has been shown to bind PVR with high affinity; this binding is thought to assist interactions between TFH and dendritic cells to regulate T cell dependent B cell responses.
  • TFH follicular B helper T cells
  • anti-TIGIT antibodies include, but are not limited to, the antibodies in WO 2016/028656 Al, WO 2017/030823 A2, WO 2017/053748 A2, WO 2018/033798 Al, WO 2017/059095 Al, and WO
  • the protein is an anti-OX40 (i.e., CD134) antibody.
  • 0X40 is a cytokine of the tumor necrosis factor (TNF) ligand family.
  • TNF tumor necrosis factor
  • 0X40 functions in T cell antigen-presenting cell (APC) interactions and mediates adhesion of activated T cells to endothelial cells.
  • anti-OX40 antibodies include, but are not limited to, WO 2018/031490 A2, WO 2015/153513 Al, WO 2017/021912 Al, WO 2017/050729 Al,
  • WO 2017/096182 Al WO 2017/134292 Al, WO 2013/038191 A2, WO 2017/096281 Al, WO 2013/028231 Al, WO 2016/057667 Al, WO 2014/148895 Al, WO
  • the protein is an anti-IL8 antibody.
  • IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus.
  • the protein is Abatacept (marketed as ORENCIA®).
  • Abatacept is a drug used to treat autoimmune diseases like rheumatoid arthritis, by interfering with the immune activity of T cells.
  • Abatacept is a fusion protein composed of the Fc region of the immunoglobulin IgGl fused to the extracellular domain of CTLA-4.
  • an antigen presenting cell In order for a T cell to be activated and produce an immune response, an antigen presenting cell must present two signals to the T cell. One of those signals is the major histocompatibility complex (MHC), combined with the antigen, and the other signal is the CD80 or CD86 molecule (also known as B7-1 and B7- 2) ⁇
  • MHC major histocompatibility complex
  • the protein is Belatacept (trade name NULOJIX®).
  • Belatacept is a fusion protein composed of the Fc fragment of a human IgGl
  • a system for controlling, modulating, increasing, or improving protein yield in a sample mixture comprising a target protein and impurities comprising monitoring in real-time an ultraviolet (UV) signal of the sample mixture during protein filtration in a harvest skid.
  • UV ultraviolet
  • Systems disclosed herein comprise one or more sensors.
  • the sensors comprise pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.
  • the harvest skid is designed to integrate all sensors
  • the system comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers.
  • the PMAT controllers are built on the cart to accommodate a total of ten different sensors.
  • a gravity pump e.g a LEVITRONIX® gravity pump
  • the system is movable, lockable and/or e-stoppable.
  • a system or device comprises the
  • a system or device comprises the embodiment of FIG. 2.
  • the apparatus may include one or more sensor.
  • the sensors may comprise pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.
  • the apparatus is designed to integrate all sensors, including pressure (4), UV (1), turbidity (2), temperature (2) and flow sensor (1), into the apparatus.
  • the apparatus comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers.
  • the PMAT controllers are built into the apparatus to accommodate a total of ten different sensors.
  • a gravity pump e.g., a LEVITRONIX® gravity pump
  • the system is movable, lockable and/or e-stoppable.
  • the apparatus may also comprise a processor configured to control collection of the target protein.
  • the processor may also be configured to change a condition of the apparatus, for example, the temperature, pressure, turbidity, or flow.
  • the processor may also be configured to control the collection of the target protein.
  • the processor may use an established model to determine a culture harvesting process.
  • the cell culture harvesting process may comprise a filtration based cell culture harvesting process.
  • the processor may be configured to use a target protein titer.
  • the apparatus may be incorporate into a system for controlling, modulating, increasing, or improving protein yield in a sample mixture comprising a target protein and impurities. d. Process
  • a system or device comprises the embodiment of FIG. 2., which demonstrates the process flow using this harvest skid.
  • Liquid from water source/bioreactor/PBS source was driven to depth filter by the LEVITRONIX® gravity pump.
  • a LEVITRONIX® flow sensor was placed after the pump.
  • Delta VTM calculated flow totalizer volume through online flow sensor readings.
  • Flow totalizer volume was used to determine the end of water flush.
  • Four pressure sensors were placed before primary depth filter, secondary depth filter, pre-filter and sterile filter. Pressure-flow control loop worked based on real-time pressure value before the primary depth filter.
  • Two turbidity sensors were placed after primary and secondary depth filters as indicators of filtrate quality.
  • One UV sensor was placed after the secondary depth filter and used to calculate online target protein concentration and control cut-off of clarified bulk collection.
  • Real-time upstream source and downstream receiving vessel weights were monitored and displayed on Delta VTM as well.
  • FIG. 1 shows a schematic of the harvest skid. This harvest skid was designed to integrate all sensors, including pressure (4), UV (1), turbidity (2), temperature (2) and flow sensor (1), into one cart. See Figure 2. Three PMAT controllers were built on the cart to accommodate a total of ten different sensors. A LEVITRONIX® gravity pump, which was used to drive liquid to depth filters, was also mounted on the skid cart. This harvest skid was designed to be movable, lockable and e-stoppable.
  • the sensors used by the harvest have distinct functions.
  • the pressure sensor monitors pressure during process; cascade control to inlet pump, reduce inlet pump flow rate when pressure is too high.
  • the UV sensor monitors UV signal after depth filtration during process; translated to protein concentration to control start and end of bulk collection. UV is measures at 280 nm.
  • the weight sensor monitors upstream bioreactor and downstream receiver weight during process; control load and chase steps. Bioreactor and receiver load cell values are integrated into harvest skid control system.
  • the turbidity sensor which measures turbidity at 880 nm, monitors turbidity before and after depth filtration during process. Turbidity breakthrough is observed if the depth filters are fouled.
  • the temperature sensor monitors temperature during process. The harvest process herein operates under ambient (room) temperature.
  • the harvest skid process was used to purify proteins of interest from cell culture.
  • Liquid from water source/bioreactor/PBS source was driven to primary depth filter by the LEVITRONIX® gravity pump.
  • LEVITRONIX® gravity pump was proven to cause less cell death in CHO cell culture than the peristaltic pumps P3P.
  • a LEVITRONIX® flow sensor was placed after the pump.
  • Delta VTM calculated flow totalizer volume through online flow sensor readings. Flow totalizer volume was used to determine the end of water flush.
  • Four pressure sensors were placed before primary depth filter, secondary depth filter, pre-filter and sterile filter P4P. Pressure-flow control loop worked based on real-time pressure value before the primary depth filter. If the pressure values exceed a certain threshold, Delta VTM would automatically drive the pump to down-regulate pump speed.
  • Two turbidity sensors were placed after primary and secondary depth filters as indicators of filtrate quality.
  • One ETV sensor value of which was used to calculate online target protein concentration and control cut-off of clarified bulk collection, was placed after the secondary depth filter.
  • Real-time upstream source and downstream receiving vessel weights were monitored and displayed on Delta VTM as well.
  • the harvest skid uses each sensor to detect values at the following ranges and accuracies.
  • the methods disclosed herein eliminated air blow-down step. Meanwhile, the start and end of collection of clarified bulk were controlled automatically based on online ETV readings and calculated titer. See Figure 3. More specifically, real-time target protein concentration during the harvest process was calculated by online UV sensor readings, using the models generated. Cut-off of bulk collection was therefore determined directly on calculated online target protein concentration. The calculation algorithms were integrated into Delta VTM control system to achieve automatic cut-off of clarified bulk collection.
  • Path-length of this UV sensor was adjusted to accommodate a total target protein concentration of 0-6 g/L in the range of 0-2 AU.
  • Other UV sensors or Flow VPE (C- technologies) could be used for higher concentration determination.
  • UV sensor path length was adjusted to cover a wide range of target protein concentrations that could be observed during the harvest process. As UV readings close to 2 AU (maximum output) are less accurate, path length was adjusted down so that UV reading was around 1.6 at titer of 5 g/L. Good linearity was observed with R 2 of 0.97. Thus, the serial dilution of a cell culture sample provides a strong correlation between UV reading and titer.
  • the depth filters were scaled down based on a loading capacity of 60 L/m 2 (per primary filter). Offline samples after secondary depth filter were collected during the harvest process. Offline titer readings were plotted with online UV sensor values to understand the relationship between pure protein concentration and online UV signal during harvest process.
  • a second small scale harvest test was carried out using 2 L of clarified bulk (eTau cell culture with cells removed) with a titer of 5 g/L.
  • the depth filters were scaled down based on a loading capacity of 60 L/m 2 (per primary filter). Offline samples after secondary depth filter were collected during the harvest process. Offline titer readings were plotted with online UV sensor values to understand the relationship between target protein concentration (in a mixture of culture components) and online UV signal during harvest process.
  • Model Predicted Titer a + b*(online UV signal).
  • Model Predicted Titer A + B*exp(C* online UV signal).
  • Figure 12 shows the difference for each process tested. Overall mean difference ranges from 0.07-0.36 g/L, suggesting the models could be reliably applied to different processes with a variety of properties as shown in Table 2.
  • yield using the new harvest skid was 2-5% higher than that using the old method.
  • a new target protein is selected to be clarified using this harvest skid.
  • All sensors on the harvest skid are connected in-line as described in FIG. 2 to monitor pressure, flow, UV, turbidity and temperature during the harvest process.
  • water source is connected with LEVITRONIX® gravity pump.
  • Total flow amount and flow rate are entered into Delta VTM to control the depth filter flushing step.
  • bioreactor source is connected with LEVITRONIX® gravity pump to start loading of cell culture to depth filters.
  • ETV prediction model constants for incline portion are entered into Delta VTM; and start of collection cut-off threshold are entered into Delta VTM. Online ETV signal is translated to target protein concentration during loading.
  • the receiving vessel is connected with the sterile filter to collect clarified bulk.
  • PBS source is connected with LEVITRONIX® gravity pump to start chasing step.
  • UV prediction model constants for decline portion are entered into Delta VTM based on cell line type; and end of collection cut-off threshold is entered into Delta VTM.
  • Online UV signal is translated to target protein concentration during chasing.
  • a harvest process started with a water-for-injection (WFI) flush of depth filters.
  • WFI water-for-injection
  • UV sensor was connected to the outlet of a secondary depth filter. Once the filtration became steady, a clear flow was seen from the outlet; the UV sensor was zeroed at that point. After the desired amount of WFI was flushed through the filter, cell culture media were connected with the filter inlet to begin the loading. The online UV trace of the media was monitored along the loading. The filtrate samples were taken along the loading and analyzed offline by a titer assay. FIG. 11 shows that the titer trace was achieved by modelling from the UV signal. The titer trace by modelling was well aligned with the offline titer assay results and therefore can be used to start and end the collection for process robustness and yield improvement.
  • the apparatus may include one or more sensor.
  • the sensors may comprise pressure sensors, UV sensors, turbidity sensors, temperature sensors, flow sensors, and any combination thereof.
  • the apparatus is designed to integrate all sensors, including pressure (4), UV (1), turbidity (2), temperature (2) and flow sensor (1), into the apparatus.
  • the apparatus comprises three PMAT (Pressure Monitor Alarm Transmitter) controllers.
  • the PMAT controllers are built into the apparatus to accommodate a total of ten different sensors.
  • a gravity pump e.g., a LEVITRONIX® gravity pump
  • the system is movable, lockable and/or e-stoppable.
  • the apparatus may also comprise a processor configured to control collection of the target protein.
  • the processor may also be configured to change a condition of the apparatus, for example, the temperature, pressure, turbidity, or flow.
  • the processor may also be configured to control the collection of the target protein.
  • the processor may use an established model to determine a culture harvesting process.
  • the cell culture harvesting process may comprise a filtration based cell culture harvesting process.
  • the processor may be configured to use a target protein titer.
  • the apparatus may be incorporate into a system for controlling, modulating, increasing, or improving protein yield in a sample mixture comprising a target protein and impurities.

Abstract

L'invention concerne des procédés de contrôle, de modulation, d'augmentation ou d'amélioration du rendement en protéines dans un mélange d'échantillons contenant une protéine cible et des impuretés comprenant la surveillance en temps réel d'un signal ultraviolet (UV) du mélange d'échantillons pendant la filtration de protéines dans une récolte skid.
PCT/US2019/024132 2018-03-27 2019-03-26 Surveillance en temps réel de la concentration de protéines à l'aide d'un signal ultraviolet WO2019191133A1 (fr)

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JP2020551818A JP2021519298A (ja) 2018-03-27 2019-03-26 紫外線シグナルを使用した力価のリアルタイムモニタリング
EP19719664.5A EP3774840A1 (fr) 2018-03-27 2019-03-26 Surveillance en temps réel de la concentration de protéines à l'aide d'un signal ultraviolet
US17/041,851 US20210024873A1 (en) 2018-03-27 2019-03-26 Real-time monitoring of protein concentration using ultraviolet signal
KR1020207030632A KR20200136454A (ko) 2018-03-27 2019-03-26 자외선 신호를 사용한 단백질 농도의 실시간 모니터링
CN201980022884.1A CN111918873A (zh) 2018-03-27 2019-03-26 使用紫外线信号实时监测滴度

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WO2021260126A1 (fr) * 2020-06-26 2021-12-30 Global Life Sciences Solutions Usa Llc Système de biotraitement et son procédé associé
WO2022251194A1 (fr) * 2021-05-25 2022-12-01 Boehringer Ingelheim International Gmbh Procédé de titrage utilisant une mesure uv pour la production biologique continue

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